Summary: Studies of this laboratory have been pivotal for understanding the interaction between CRH and vasopressin (VP) in the regulation of pituitary ACTH, and the regulation of the expression of these peptides in the PVN during stress and other alterations of the HPA axis. Both peptides co-expressed in the same parvocellular neuron of the paraventricular nucleus (PVN) are differentially regulated during stress or exposure to glucocorticoids. CRH coordinates behavioral, autonomic and hormonal responses to stress and is the main regulator of ACTH secretion in acute and chronic conditions. Following CRF release, activation of CRF transcription is required to restore mRNA and peptide levels, but termination of the response is essential to prevent pathology associated with chronic elevation of CRF and glucocorticoid production. While glucocorticoid feedback plays an important role in regulating CRF expression, the relative importance of direct transcriptional repression of the CRF gene by glucocorticoids in the overall feedback mechanism is not clear. In addition to glucocorticoids, intracellular feedback mechanisms in the CRF neuron, involving induction of repressor forms of cAMP response element modulator (CREM) limit CRF transcriptional responses by competing with the positive regulator, phospho-CREB. Research during the past year supports the hypothesis that induction of ICER is part of an intracellular feedback mechanism limiting CRH transcription. In these studies inhibition of endogenous ICER production using silencing RNA (siRNA) enhanced forskolin-induced CRH promoter activity in reporter gene assays, and attenuated the inhibitory phase of CRH transcription in primary hypothalamic neuronal cultures incubated with forskolin. Rapid repression of CRF transcription following stress-induced activation is likely to contribute to limiting the stress response and to preventing disorders associated with excessive CRF production.[unreadable] [unreadable] While it is well established that cAMP-dependent signaling is the major regulator of CRH transcription, the most recognized receptors mediating stimulation of CRH neurons, alpha adrenergic and glutamaergic receptors, act through mechanisms other than via cAMP. To determine whether synergism between calcium phospholipid signaling and small elevations of intracellular cAMP mediate activation of CRH transcription during stress, we investigated the effects of the phorbol ester, PMA, and the cAMP stimulator, forskolin, on CRH transcription and CREB phosphorylation in the hypothalamic cell line, 4B, and in hypothalamic neuronal cultures. Incubation of 4B cells transfected with a CRH promoter-driven reporter with forskolin, increased phospho-CREB (pCREB) levels and CRH promoter activity. PMA alone increased pCREB to similar levels but had no effect on CRH promoter activity. However, PMA potentiated the stimulatory effect of small concentrations of forskolin. The CREB dominant negative, A-CREB, reduced forskolin-stimulated CRH promoter activity in the absence or presence of PMA, indicating that pCREB is required but not sufficient to activate CRH transcription. PMA also potentiated the stimulatory effect of forskolin on CRH transcription (measured by intronic qRT-PCR) in primary cultures of hypothalamic cells. The potentiating effect of calcium/phospholipid-signaling on cAMP-mediated transcription provides a mechanism by which non-cAMP dependent neurotransmitters mediate activation of CRH transcription in presence of minor increases in intracellular cAMP. The relevance of these observations to the regulation of CRH expression by neurotransmitters activated during stress is under current investigation. [unreadable] [unreadable] We have shown that stress activates the renin-angiotensin system and increases the number of type angiotensin (AT1) receptors in the PVN. Sustained pretreatment with AT1 receptor antagonists prevents the sympathoadrenal and hormonal responses to 24 hours isolation stress. To examine the mechanism of the anti-stress effects of AT1 receptor antagonism, we investigated the effect of chronic treatment subcutaneous infusion of candesartan, a non-competitive AT1 receptor antagonist, on HPA axis responses to 24h isolation stress in Wistar rats. In vehicle rats, 24 h isolation stress increased pituitary ACTH, adrenal corticosterone content and AT1 receptor binding in the PVN but decreased CRH mRNA and CRH content in the PVN, changes similar to those found in depression. Candesartan pretreatment prevented the effects of stress, suggesting that activation of AT1 receptors is permissive for the HPA axis response to isolation. These results support the view that inhibition of central AT1 receptors limits the CRF response to stress and could serve as a therapeutic tool to preserve homeostasis under stress chronic conditions.